Multiple sclerosis (MS) is a common and disabling disease of the central nervous system (CNS). While MS was formally believed to be a demyelinating disease, it is now clear that axonal degeneration occurs prominently in MS and is the primary cause of permanent disability in MS. The pathogenesis of axonal degeneration in MS is imprecisely understood but activated microglia are believed to be involved in axonal destructions at all stages of MS. Lipoic acid is an effective therapy for the murine model of MS, experimental autoimmune encephalomyelitis (EAE), and inhibits T cell migration into the CNS in EAE. The mechanism of action of lipoic acid appears to involve its ability to stimulate the production of the small second molecule messenger cAMP via the prostaglandin G-coupled protein receptors EP2 and EP4. Preliminary evidence suggests that lipoic acid may inhibit microglial activation independent of its effects on T cells and may do so via stimulation of cAMP production. This research project will test the hypothesis that lipoic acid stimulates cAMP production in microglia cells via the EP2 and EP4 receptors and thereby prevents microglial activation and release of soluble inflammatory mediators that damage axons. This will be accomplished in the work described in the three Specific Aims. For Specific Aim 1, microglia cultured from mouse brains will be activated using either lipopolysaccharide (LPS) or the cytokines tumor necrosis factor-alpha (TNF-1) and interferon-gamma (IFN-3) in the presence or absence of varying concentrations of lipoic acid. Microglial activation will be measured by assessing production of nitric oxide (NO), inflammatory cytokines, and expression of cell surface activation markers. This will determine whether lipoic acid can inhibit microglial activation in vitro. The ability of lipoic acid to stimulate cAMP production in cultured microglial cells will be determined by live cell fluorescent imaging using a FRET-based technology and a cAMP reporter. To determine whether inhibition of microglial activation by lipoic acid is dependent on cAMP production, an inhibitor of cAMP will be tested for its ability to reverse inhibition of microglial activation by lipoic acid. Finally, to determine the dependence of the effects of lipoic acid on the EP2 and EP4 receptors, the ability of lipoic acid to inhibit activation of microglia EP2 and EP4 knock-out mice will be compared with that of wild type microglia. For Specific Aim 2, microglial activation will be induced in vivo by intracerebral injection of TNF-1/IFN-3 or LPS and the ability of systemically administered lipoic acid to block microglial activation will be determined. Measures of in vivo microglial activation will include number of CD11b+ microglial, upregulation of the cell surface expression of MHC Class II, galectin-3 and iNOS determined by quantitative immunofluorescence. Experiments will be repeated using EP2 knock-out and EP4flox/flox/CD11bCre conditional knock-out mice and results compared with those obtained in wild type mice to determine the dependence of the inhibitory effects of lipoic acid on the EP2 and EP4 receptors on microglia. For Specific Aim 3, the therapeutic effects of lipoic acid on EAE in wild type and EP2 knock-out and EP4flox/flox/CD11bCre conditional knock-out mice will be compared to determine whether the mechanism of action of lipoic acid depends on the EP2 and EP4 receptors. The results of this research will provide further insights into the mechanism of action of lipoic acid in EAE and provide additional pre-clinical data needed for guiding the development of lipoic acid as a treatment for MS. PUBLIC HEALTH RELEVANCE: This research project is relevant to veterans'health and healthcare issue because its purpose is ultimately to develop lipoic acid as a new and cost effective treatment for multiple sclerosis (MS). MS is an important neurologic disease among veterans. A report on VHA health care utilization prepared by the VA MS Centers Excellence revealed that over 24,000 veterans with MS received care through VHA from 2000-2003 (MS Center of Excellence, 2004 http://www.va.gov/ms). Over 6,000 of these individuals were service connected for MS and over 9,000 were taking one of the disease modifying drugs. The average annual cost to VHA for providing care to veterans with MS exceeded $35,000 per patient per year. In addition, many veterans with MS become disabled and are eligible for VA pensions, providing another source of tremendous expense. Current FDA approved therapies for MS cost up to $35,000 a year and are only partially effective. Finding new, cost effective therapies that can slow the progression of MS is of obvious relevance to the health care mission of VHA.